5 5 dimethyl 1 pyrroline n oxide dmpo

Manufactured by Dojindo Laboratories

Sourced in Japan, China

5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a chemical compound that serves as a spin trapping agent. Spin trapping is a technique used in electron paramagnetic resonance (EPR) spectroscopy to detect and analyze short-lived free radicals. DMPO can form stable adducts with these free radicals, allowing for their identification and quantification.

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5,5-dimethyl-1-pyrroline N-oxide (13 citations) Purified 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (2 citations)

19 protocols using 5 5 dimethyl 1 pyrroline n oxide dmpo

Synthesis and Characterization of Dunnione Derivatives

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Dunnione derivatives (DUNs) were synthesized as described by Bian et al. [14 (link)]. They were more than 95% pure, as demonstrated by NMR and mass spectrometry analyses.
The [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434) was prepared as described by Boutin et al. [17 (link)], and menadione, Dulbecco’s phosphate buffered saline, Tris-HCL, octyl β-d-glucopyranoside were from Sigma-Aldrich (Saint Quentin Fallavier, France). 5,5′-Dimethyl-1-pyrroline N-oxide (DMPO) was from Dojindo (Kumamoto, Japan). N-benzyldihydro-nicotinamide (BNAH) was from TCI Europe (Zwijndrecht, Belgium).

Chhour M., Aubouy A., Bourgeade-Delmas S., Pério P., Ternet-Fontebasso H., Haidara M., Ferry G., Nepveu F., Boutin J.A, & Reybier K. (2019). Antimalarial Properties of Dunnione Derivatives as NQO2 Substrates. Molecules, 24(20), 3697.

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Biochemical Analysis of α-Synuclein Pathology

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Cytochrome c, Maneb (Manganese ethylene-1,2-bisdithiocarbamate), paraquat (1,1-dimethyl-4,4-bipyridinium), paraformaldehyde, recombinant α-synuclein, Triton X-100, and TRI Reagent were from Sigma (St. Louis, MO). 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Dojindo Laboratories (Rockville, MD) and used without further purification. Chicken polyclonal and mouse monoclonal anti-DMPO antibodies were developed in our laboratory and used in the immuno-spin trapping studies. Rabbit polyclonal anti-tyrosine hydroxylase antibody was from Millipore (Billerica, MA). Rabbit anti-α-synuclein, mouse anti-cytochrome c, anti-complex IV, anti-caspase-3, anti-caspase-9 and anti-β-actin antibodies were from Abcam (Cambridge, MA). Micro BCA Protein Assay Kit, Permount, RIPA buffer, Surfact-Amps X-100, Mitochondria Isolation Kits for Tissue, and Pierce Classic Immunoprecipitation Kits were from Thermo Scientific (Rockford, IL). Nitrocellulose membranes, Prolong Gold anti-fade reagent with DAPI and AlexaFluor secondary antibodies were from Invitrogen (Grand Island, NY). The Vectastain Elite ABC kit for immunohistochemistry was from Vector Laboratories (Peterborough, UK). RNeasy mini kit columns were from Qiagen (Valencia, CA). Agilent Whole Mouse 4X44K microarray slides and the reagents used in microarray experiments were all from Agilent Technologies (Santa Clara, CA).

Kumar A., Ganini D, & Mason R.P. (2016). Role of cytochrome c in α-synuclein radical formation: implications of α-synuclein in neuronal death in Maneb- and paraquat-induced model of Parkinson’s disease. Molecular Neurodegeneration, 11, 70.

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Measuring Reactive Oxygen and Nitrogen Species

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The concentrations of H₂O₂ and NO₂⁻/NO₃⁻ in the water were measured using a hydrogen peroxide/peroxidase assay kit (Thermo Fisher Scientific) and a nitrite/nitrate colorimetric assay kit (Cayman), respectively. ˙OH, ¹O₂, ˙NO, O₂˙⁻, ˙NO₂, and ONOO⁻ were measured using an electron spin resonance (ESR) spectroscope (Bruker) with relevant spin traps (29 (link)). The spin traps were 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Dojindo) for trapping ˙OH, 5 mM N-(dithiocarbamoyl)-N-methyl-d-glucamine (MGD; Dojindo) for trapping ˙NO, 10 mM 2,2,6,6-tetramethylpiperidine (TEMP; TCI) for trapping ¹O₂, and 10 mM 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONE-H; Enzo) for trapping O₂˙⁻, ˙NO₂, and ONOO⁻.

Guo L., Xu R., Gou L., Liu Z., Zhao Y., Liu D., Zhang L., Chen H, & Kong M.G. (2018). Mechanism of Virus Inactivation by Cold Atmospheric-Pressure Plasma and Plasma-Activated Water. Applied and Environmental Microbiology, 84(17), e00726-18.

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Immunostaining and Oxidative Stress Assays

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Apocynin, bovine serum albumin, N-3-(aminomethyl)benzylacetamide ·2HCl (1400W), Maneb (Manganese ethylene-1,2-bisdithiocarbamate), DAB, NADPH, paraquat ( 1,1-dimethyl-4,4-bipyridinium), paraformaldehyde, Triton X-100 , and 5,10,15,20,-tetrakis(4-sulfonatophenyl) porphyrinato iron (III) chloride (FeTPPS) were from Sigma (St. Louis, MO). 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Dojindo Laboratories (Rockville, MD) and used without further purification. Chicken and rabbit polyclonal anti-DMPO antibodies were developed in our laboratory and used in the immuno-spin trapping studies. Mouse monoclonal anti-integrin □M (ox-42), rabbit polyclonal anti-iNOS, and P67 phox antibodies were from Santa Cruz Biotechnology (Dallas, TX). Mouse monoclonal anti-alpha synuclein, and anti-β-actin antibodies were from Abcam (Cambridge, MA). Rabbit polyclonal anti-tyrosine hydroxylase antibody was from Millipore (Billerica, MA). Micro BCA Protein Assay Kit, Permount, RIPA buffer, Surfact-Amps X-100, and Pierce Classic Immunoprecipitation Kits were from Thermo Scientific (Rockford, IL). Nitrocellulose membranes, Prolong Gold anti-fade reagent with DAPI and AlexaFluor secondary antibodies were from Invitrogen (Grand Island, NY). The Vectastain Elite abc kit was from Vector laboratories (Peterborough, UK).

Kumar A., Leinisch F., Kadiiska M.B., Corbett J, & Mason R.P. (2015). Formation and implications of alpha-synuclein radical in Maneb- and paraquat-induced models of Parkinson’s disease. Molecular neurobiology, 53(5), 2983-2994.

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Radical Scavenging Assay Reagents

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5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) and bis-(2-hydroxyethyl)-iminotris-(hydroxymethyl)-methane (Bis-Tris) were purchased from Dojindo Laboratories, Ltd. (Kumamoto, Japan). Dimethyl sulfoxide (DMSO) and 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Manganese (IV) oxide (MnO₂: >70%) was purchased from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan). 4-Hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL) and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Ammonia solution (28–30%), H₂O₂ (30.0–35.0%), and glutathione (GSH) were purchased from Wako Pure Chemical Industries, Ltd. (Fujifilm Wako Pure Chemical Corp., Osaka, Japan). Cerium nitrate hexahydrate was purchased from Nikki Corp., Saitama, Japan. Deionized water (Milli-Q system; Merck Millipore, Billerica, MA) was used for all experiments.

Ogawa Y., Kawaguchi T., Tanaka M., Hashimoto A., Fukui K., Uekawa N., Ozawa T., Kamachi T, & Kohno M. (2023). Quenching effect of cerium oxide nanoparticles on singlet oxygen: validation of the potential for reaction with multiple reactive oxygen species. Journal of Clinical Biochemistry and Nutrition, 73(1), 1-8.

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Intracellular ROS Generation Measured by ESR

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Intracellular ROS generation after iron treatment was detected by electron spin resonance (ESR) according to a previous study.^{(24 (link))} Briefly, cells were seeded on a sterilized glass cover slide (49 × 5 × 0.2 mm) at confluency and incubated overnight. Cells were then exposed to fresh medium containing 500 µM FeSO₄ for 1 h and then immersed in the respiratory buffer containing 5 mM succinate (Sigma-Aldrich Japan K.K., Tokyo, Japan), 5 mM malate (Wako), 5 mM glutamate (Sigma-Aldrich Japan K.K.), 5 mM nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich Japan K.K.) and 5 µl 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Dojindo Laboratories, Kumamoto, Japan). The slide was set in a tissue glass, and ESR spectra were obtained using a JEOL-TE X-band spectrometer (JEOL, Ltd., Tokyo, Japan) using the following measurement conditions: 10 mW incident microwave power, 9.4 GHz frequency, and 0.1 mT field modulation amplitude. The experiments were performed three times independently.

Ito H., Kurokawa H., Hirayama A., P. Indo H., J. Majima H, & Matsui H. (2017). Cancer cell-specific mitochondrial reactive oxygen species promote non-heme iron uptake and enhance the proliferation of gastric epithelial cancer cell. Journal of Clinical Biochemistry and Nutrition, 61(3), 183-188.

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Synthesis and Preparation of DMPO and MCP

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5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Dojindo Laboratories, Ltd. (Kumamoto, Japan). 3-Methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (MCP, also called MC-PROXYL) was synthesized according to previously reported methods.^{(20 (link))} MCP was prepared as a 150 mM i.v. injectable isotonic solution (of sucrose). Other chemicals were of analytical grade. Deionized water (deionization by the Milli-Q system) was used for all experiments.

Ueno M., Shibata S., Nakanishi I., Aoki I., Yamada K.I, & Matsumoto K.I. (2022). Effects of selenium deficiency on biological results of X-ray and carbon-ion beam irradiation in mice. Journal of Clinical Biochemistry and Nutrition, 72(2), 107-116.

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DMPO-Mediated Oxidative Stress Assay

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5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Dojindo Laboratories, Ltd. (Kumamoto, Japan). TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) was purchased from Sigma-Aldrich Co. (St. Louis, MO). GSH and 30% H₂O₂ solution was purchased from Wako Chemical Co. (Tokyo, Japan). Other chemicals were of analytical grade. As the basic solvent for the reaction mixtures, 100 mmol/L phosphate buffer containing 0.05 mmol/L diethylenetriaminepentaacetic acid (DTPA) was prepared at pH 7.0 and used for all experiments. Deionized water (Milli-Q system, Merck Millipore, Billerica, MA) was used for preparing the 100 mM phosphate buffer.

Matsumoto K.I., Nyui M., Ueno M., Ogawa Y, & Nakanishi I. (2019). A quantitative analysis of carbon-ion beam-induced reactive oxygen species and redox reactions. Journal of Clinical Biochemistry and Nutrition, 65(1), 1-7.

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ROS Quantification via ESR Spectroscopy

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ROS generation in cells was measured using electron spin resonance (ESR) according to previous study.^{(13 (link))} Cells were seeded on a glass cover slide (49 × 5 × 0.2 mm) and incubated overnight. Cells were exposed to the medium containing 1 mM IND for 1 h. Cells were immersed in respiratory buffer containing 5 mM succinate (Sigma-Aldrich Japan K.K., Tokyo, Japan), 5 mM malate (Wako Pure Chem. Ind., Ltd., Osaka, Japan), 5 mM glutamate (Sigma-Aldrich Japan K.K.), 5 mM nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich Japan K.K.), and 10 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (DOJINDO). The cell-attached glass cover slide was placed on a tissue glass, and the ESR spectra were obtained by inserting the tissue glass into the device. All ESR spectra were obtained using a JEOL-TE X-band spectrometer (JEOL Ltd., Tokyo, Japan) under the following conditions: 20 mW incident microwave power, 9.42 GHz frequency, and 0.1 mT field modulation amplitude.

Ito H., Matsui H., Hirayama A., Indo H.P., Majima H.J, & Hyodo I. (2016). Reactive oxygen species induced by non-steroidal anti-inflammatory drugs enhance the effects of photodynamic therapy in gastric cancer cells. Journal of Clinical Biochemistry and Nutrition, 58(3), 180-185.

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DMPO, TEMPOL, and Caffeine Preparation

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5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Dojindo Laboratories, Ltd. (Kumamoto, Japan). 4-Hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) and caffeine were purchased from Sigma-Aldrich (St. Louis, MO). Deionized water (deionization by the Milli-Q system, Merck Millipore, Billerica, MA) was used for all sample preparations.

Ogawa Y., Sekine-Suzuki E., Ueno M., Nakanishi I, & Matsumoto K.I. (2018). Localized hydroxyl radical generation at mmol/L and mol/L levels in water by photon irradiation. Journal of Clinical Biochemistry and Nutrition, 63(2), 97-101.

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